The ability now exists to amplify a DNA sequence without it being necessary to clone it in a host cell. The best known technique in this field is the so-called polymerase chain amplification technique, often designated by the acronym PCR, described, for example, in U.S. Pat. Nos. 4,683,195 and 4,683,202.
The PCR amplification method may be combined with a prior step of reverse transcription in order to amplify an RNA sequence in the form of DNA.
The polymerase chain amplification reaction coupled to reverse transcription (often designated in abbreviated form by the acronym RT-PCR) is now widely used to monitor gene expression at messenger RNA (mRNA) level, as well as for the direct cloning of coding sequences.
The polymerase chain amplification reaction coupled to reverse transcription hence consists in carrying out the synthesis of a first strand of complementary DNA (cDNA) using a reverse transcriptase, in denaturing the RNA-cDNA heteroduplex formed, in synthesizing a second cDNA strand, complementary to the first strand, under the action of a DNA polymerase, and in then subjecting the double-stranded cDNA obtained to a polymerase chain amplification according to known methods, which consist of a series of successive reactions of denaturation of the double-stranded DNA followed by its replication under the action of a DNA polymerase.
More specifically, the synthesis of the first cDNA strand is carried out using nucleoside triphosphates, by elongation of an oligonucleotide primer under the action of a reverse transcriptase. Since the copying of the RNA template takes place in the 3'A5' direction of the template, the oligonucleotide primer must be capable of hybridizing with a sequence located in the vicinity of the 3' end of the RNA to be copied. For this reason, the primer used in the reverse transcription operation is referred to as a 3' primer.
To carry out the synthesis of the second complementary DNA strand, the procedure is to synthesize the elongation product of a second primer capable of hybridizing with a sequence adjoining the 3' end of the first cDNA strand. In other words, the second primer must be identical to a sequence adjoining the 5' end of the starting RNA, or must be sufficiently homologous with such a sequence to be capable of hybridizing with said sequence of the first cDNA strand. For this reason, this second primer is referred to as a 5' primer.
The sequence to be amplified is hence flanked by the two primers.
Regardless of the methodology used, the RT-PCR reaction entails several successive reaction steps, namely denaturation of the RNA, hybridization of the 3' primer with the RNA, synthesis of a first cDNA strand using an enzyme possessing RNA-dependent DNA polymerase (or reverse transcriptase) activity, denaturation of the RNA-cDNA heteroduplex formed, hybridization of the 5' primer with the first cDNA strand, synthesis of the second cDNA strand using an enzyme possessing DNA-dependent DNA polymerase activity, and then the succession of amplification reactions by denaturation of the double-stranded DNA, hybridization of the 3' and 5' primers with the strand to which they are respectively complementary and synthesis of the second DNA strands by elongation from the primers.
The RT-PCR method is very sensitive. This great sensitivity is, moreover, often the source of "false-positive" results, caused by residual traces of genomic DNA in the mRNA preparations. For similar reasons, it is very important to avoid the accidental introduction of contaminating sequences. It is hence desirable to reduce to a minimum the risks of contamination during the succession of reactions constituting the chain amplification. However, depending on the methodology used, various manipulations may be required during the reaction, increasing the risk of introduction of contaminating sequences.
In its least sophisticated implementation, the RT-PCR method entails three steps, namely:
1) denaturation of the RNA by heating; PA1 2) synthesis of the first cDNA strand in a buffer containing, apart from the nucleoside triphosphates, a 3' primer and a reverse transcriptase; and PA1 3) synthesis of the second cDNA strand by addition of the 5' primer and a DNA polymerase, followed by the succession of amplification reactions by PCR; see, for example, Stefan SCHWARTZ, Journal of Virology, Vol. 64, No. 6, Jun. 1990, pp. 2519-259. PA1 obtaining a starting solution containing the RNA of said sample in a suitable buffer, PA1 denaturing the RNA contained in said solution by heating, PA1 treating the solution obtained, containing the denatured RNA, with a first primer (or 3' primer) under conditions permitting hybridization of the primer followed by the synthesis of a first DNA strand complementary to the RNA sequence to be amplified, in the presence of a sufficient quantity of an enzyme system having reverse transcriptase activity in said buffer, PA1 denaturing the RNA-cDNA heteroduplex formed, and treating the solution obtained with a second primer (or 5' primer) under conditions permitting hybridization of the second primer followed by the synthesis of the second cDNA strand, in the presence of a heat-stable enzyme system having DNA polymerase activity in the same buffer, PA1 and subjecting the cDNA obtained to a sufficient number of amplification cycles to obtain the desired degree of amplification, wherein said solution contains, right from the start, all the reactants and solvents needed for said steps, wherein all the steps are performed in the same container and without addition of reactants or solvents, and wherein the denaturation of the RNA is performed at a temperature of not less than approximately 60.degree. C. PA1 comprising the sequence EQU CCTATCTGTCCCCTCAGCTAC (SEQ ID NO: 1) PA1 which can be used, in particular, as a 3' primer, PA1 or comprising the sequence EQU TCTATCAAAGCAACCCAC (SEQ ID NO: 2) PA1 which can be used, in particular, as a 5' primer.
With the aim of simplifying the method and of reducing the number of manipulations, a simplified method has been described, based on the discovery of a common buffer for the reverse transcriptase and for Taq DNA polymerase. This simplified method consists: 1) in denaturing the RNA in aqueous solution, the two primers, 3' and 5', already being present, and 2) in adding the ingredients of the buffer, the reverse transcriptase and the DNA polymerase to the aqueous solution. Then, without subsequent addition of reactant, successive operations of synthesis of the first cDNA strand, denaturation of the RNA-cDNA duplex, synthesis of the second cDNA strand and amplification cycles are carried out at the various appropriate temperatures; see, in particular, C. GOBLET et al., Nucl. Acids Res., 17 (5), 2144 (1989). This method is improperly referred to as a "one-step method" whereas, in fact, as just seen, it entails two steps, since the denaturation and hybridization reaction is performed before adding the ingredients needed for the steps of reverse transcription and synthesis of double-stranded DNA, thereby increasing, just as in the conventional three-step method, the risk of introducing contaminating sequences.
Another technique has been developed, consisting in using a heat-stable enzyme (rTth) possessing both RNA-dependent DNA polymerase (reverse transcriptase) activity and RNA-dependent DNA polymerase activity. However, the first activity requires the presence of manganese ions while the second requires the presence of magnesium ions see MYERS T. W. et al., Biochem. 30, 7661-7666 (1991). It is hence necessary to perform the synthesis of the first cDNA strand in the presence of MnCl.sub.2, followed by dilution of the sample with introduction of the 5' primer, MgCl.sub.2 and a chelating agent which has the power to chelate manganese ions strongly. Here too, a process of dilution with introduction of reactants necessarily follows the synthesis of the cDNA, thereby increasing the risk of introducing contaminating sequences.